Oxytocin receptor antagonist therapy in the luteal phase for implantation and pregnancy in women undergoing assisted reproductive technologies

ABSTRACT

The present invention relates to the use of an oxytocin receptor antagonist in females undergoing embryo transfer as part of an assisted reproductive technology. In particular, methods are provided for increasing ongoing implantation rate, increasing ongoing pregnancy rate, increasing clinical pregnancy rate, and/or increasing live birth rate in a female subject undergoing embryo transfer. Specifically, the antagonists are released in the luteal phase when the endometrium is receptive for embryo implantation and/or when the embryo has reached the blastocyst-stage.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of EP Application No.14199709.8, filed Dec. 22, 2014, the contents of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the use of an oxytocin receptorantagonist in females undergoing embryo transfer as part of an assistedreproductive technology. In particular, methods are provided forincreasing ongoing implantation rate, increasing ongoing pregnancy rate,increasing clinical pregnancy rate, and/or increasing live birth rate ina female subject undergoing embryo transfer. Specifically, theantagonists are released in the luteal phase when the endometrium isreceptive for embryo implantation and/or when the embryo has reached theblastocyst-stage.

BACKGROUND OF THE INVENTION

In vitro fertilization (IVF) is a method for establishing pregnancy in afemale subject. The procedure typically involves ovarian stimulationwith one or various hormones, mainly follicle-stimulating hormone (FSH),and human chorionic gonadotropin (hCG) is usually administered totrigger final follicular maturation. Oocyte retrieval takes placegenerally 2 days (around 36 h) after hCG administration. The ooctyes arethen fertilized in vitro, cultured for several days, and are transferredinto the uterus. IVF also encompasses the transfer of embryosoriginating from the eggs of a first female (the donor) into a secondfemale (the gestational carrier). Embryos may be placed in frozenstorage and transferred (i.e., frozen embryo transfer) after severalmonths or even years.

Improving the implantation rate of transferred embryos is one of themajor challenges in assisted reproductive technologies (ART) treatment.Approximately only one-third of the transferred embryos implant in womenundergoing controlled ovarian stimulation for IVF/intracytoplasmic sperminjection (ICSI). Implantation and pregnancy rates are influenced bymultiple factors related to the age and other characteristics of thepatient, the magnitude of the response to ovarian stimulation, thequality of the embryos obtained, the endometrial receptivity as well asthe actual transfer procedure.

Uterine contractility is considered a potentially important factoraffecting implantation and pregnancy rates in IVF/ICSI cycles (Fanchinet al. 1998; Schoolcraft et al. 2001; Bulleti and de Ziegler 2005). Ahigh frequency of uterine contractions at the time of transfer appearsto have a negative impact on outcome, possibly by expelling the embryosin the uterine cavity or by displacing the embryos and thereby reducingimplantation and pregnancy rates.

Observational data indicated a decrease in clinical pregnancy rates withincreasing frequency of contractions at the time of cleavage-stageembryo transfer at hCG+4 days (i.e., four days after hCG administration,corresponding to day 2 post-retrieval of oocytes) (Fanchin et al. 1998).A prospective controlled study also observed that patients with a higherfrequency of uterine contractions on the day of cleavage-stage embryotransfer (day 3 post-retrieval) had lower pregnancy rates than patientswith lower frequency of uterine contractions at the time of transfer(Zhu et al. 2014).

Uterine contractility in controlled ovarian stimulation cycles has beencompared to normal menstrual cycles (Ayoubi et al. 2003). The frequencyof uterine contractions was found to be similar between the timepoint ofhCG administration in a controlled ovarian stimulation cycle and at thetime of luteinizing hormone (LH) surge in a natural cycle. In the lutealphase, the frequency of uterine contractions was higher at hCG+4 days(corresponding to day 2 post-retrieval) in a controlled ovarianstimulation cycle compared to at LH+4 days (i.e., four days after LHsurge) in a natural cycle (Ayoubi et al. 2003). However, the frequencyof uterine contractions at LH+6 days and hCG+6 days (corresponding today 4 post-retrieval) was not different and in both situations was low,indicating identical level of uterine quiescence at that time point incontrolled ovarian stimulation and natural cycles (Ayoubi et al. 2003).In another study, uterine contractility was assessed at the day of hCGadministration, hCG+4 days (corresponding to day 2 post-retrieval) andhCG+7 days (corresponding to day 5 post-retrieval) in women undergoing acontrolled ovarian stimulation cycle (Fanchin et al. 2001). Thefrequency of uterine contractions was highest at the day of hCGadministration, decreased slightly during the early luteal phase asassessed at hCG+4 days, and reached nearly quiescent status at hCG+7days (corresponding to day 5 post-retrieval). Another study reported adecrease in the number of junctional zone contractions in oocyte donorsin the early luteal phase from day 2 to day 3 and also to day 4post-retrieval (Lesny et al. 1999). Similarly, evaluation of uterinecontractility in oocyte donors who had undergone controlled ovarianstimulation and received exogenous progesterone luteal phasesupplementation indicated that there was a significant decrease in thefrequency of uterine contractions from day 2 post-retrieval to day 5post-retrieval (Blockeel et al. 2009).

The highest level of uterine contractility is at the end of controlledovarian stimulation (day of hCG administration) and has been attributedto the high serum estradiol and low serum progesterone concentrations atthat time point. The decrease in uterine contractility during the lutealphase is believed to be the result of the exposure to endogenousprogesterone caused by the corpus luteum function in response to the hCGadministration as well as exogenous progesterone luteal supplementationused in IVF/ICSI cycles. Although progesterone supplementation is usedfor luteal phase support in IVF/ICSI patients and can reduce uterinecontractility, there is elevated uterine activity during the earlyluteal phase (day 2 or 3 post-retrieval) when transfer of cleavage-stageembryos is performed.

As uterine contractility is elevated during the early luteal phase (day2 or 3 post-retrieval) when transfer of cleavage-stage embryos isperformed, investigations assessing the impact of differentinterventions on uterine contractility for improving implantation havebeen conducted in the early luteal phase (day 2 and 3 post-retrievaltransfer; hCG+4 days). Randomized controlled trials (Moon et al. 2004;Bernabeu et al 2006; Kim et al. 2008; Ng et al. 2014), quasi-randomizedcontrolled trials (Moraloglu et al. 2010), retrospective studies infresh and frozen embryo replacement cycles (Chou et al. 2011; Lan et al.2012), or case studies in fresh and frozen embryo replacement cycles(Pierzynski et al. 2007; Liang et al 2009) reporting findings withcompounds reducing uterine contractility, like atosiban (Kim et al 2008;Moraloglu et al. 2010; Ng et al. 2014), indomethacin (Bernabeu et al.2006) and piroxicam (Moon et al. 2004) have been all conducted on day 2or 3 post-retrieval, i.e., at the time of cleavage-stage embryotransfer.

A recent randomized controlled trial (Ng et al. 2014) compared thetreatment outcome after administration of atosiban or placebo inIVF/ICSI patients followed by cleavage-stage embryo transfer on day 2 orday 3 post-retrieval. This large study was designed to determine whetherthe anecdotal evidence found in the previous smaller studies could beconfirmed. This adequately-designed, large (N=800), double-blind,randomized, controlled trial found no significant increase inimplantation or live birth rates with atosiban compared to placebo, asillustrated by live birth rates of 39.8% versus 38.0%, respectively (Nget al. 2014). Atosiban administration on day 2 or day 3 post-retrievaltherefore does not significantly increase implantation or live birthrates.

Consequently, improving implantation of transferred embryos remains oneof the major challenges in assisted reproductive technologies (ART)treatment. It is an object of the present disclosure to improveimplantation rates, thereby increasing pregnancy rates and live birthrates.

SUMMARY OF THE INVENTION

One aspect of the disclosure provides an oxytocin receptor antagonistfor use in increasing ongoing implantation rate, increasing ongoingpregnancy rate, increasing clinical pregnancy rate, and/or increasinglive birth rate, relative to control, in a female subject undergoingembryo transfer as part of an assisted reproductive technology, whereinthe antagonist is provided to the female such that the effect of theantagonist is present when the female is in (or otherwise overlaps with)the receptive endometrium stage and/or when the effect of the antagonistcoincides with the embryo reaching the blastocyst-stage. Preferably, theantagonist is provided such that it is released in the receptiveendometrium stage and/or when the embryo has reached theblastocyst-stage. Preferably, the antagonist is administered when thefemale is in the receptive endometrium stage and/or when the embryo hasreached the blastocyst-stage. In certain embodiments, the antagonist isformulated for immediate release. In other embodiments, the antagonistis formulated as a sustained or delayed release formulation, such as adepot, and is administered prior to the receptive endometrium stageand/or when the embryo is still in cleavage stage such that theantagonist is released or continues to be released once the receptiveendometrium stage is reached and/or the embryo has reached theblastocyst stage.

In preferred embodiments, the receptive endometrium stage correspondsto:

a) between LH+6 days and LH+9 days, preferably between LH+6 days andLH+8 days, most preferably on day LH+7 of a natural ovulation cycle;

b) between hCG+6 days and hCG+9 days, preferably between hCG+6 days andhCG+8 days; most preferably on day hCG+7 of an induced ovulation cycle;

c) between day 4 and day 7, preferably between day 4 and day 6, morepreferably on day 5 or 6, most preferably on day 5, of luteal phasesupport, wherein luteal phase support begins the day following oocyteretrieval in an IVF cycle, preferably wherein the female has undergoneovarian stimulation; or

d) between day 4 to day 9, preferably between day 5 to day 7, morepreferably on day 5 or day 6 of luteal phase support, preferably inpreparation for frozen embryo transfer or third party IVF, andpreferably wherein luteal support begins after the endometrium is primedfor at least 6 days with exogenous oestrogen.

Preferably, luteal phase support comprises supplementation withprogesterone, human chorionic gonadotropin, estradiol and progesterone,progestins and/or gonadatropin releasing hormone (GnRH) agonists.

Accordingly, the disclosure provides oxytocin receptor antagonists whichcan be used to prepare medicaments for increasing ongoing implantationrate, increasing ongoing pregnancy rate, increasing clinical pregnancyrate, and/or increasing live birth rate, in a female subject undergoingembryo transfer as part of an assisted reproductive technology. Alsoencompassed by the disclosure are uses of oxytocin receptor antagonistfor the preparation of a medicament for use in a female undergoingtransfer of a blastocyst-stage embryo. Preferably a medicament isadministered such that its effect overlaps with the receptiveendometrium stage and/or when the embryo has reached theblastocyst-stage. Preferably, an antagonist in a medicament is releasedin the female when the female is in the receptive endometrium stageand/or when the embryo has reached the blastocyst-stage.

The disclosure further encompasses methods for increasing ongoingimplantation rate, increasing ongoing pregnancy rate, increasingclinical pregnancy rate, and/or increasing live birth rate, in a femalesubject undergoing embryo transfer as part of an assisted reproductivetechnology, comprising administering to the female an oxytocin receptorantagonist such that the effect of the antagonist overlaps with thereceptive endometrium stage and/or when the embryo (e.g., transferredembryo) has reached the blastocyst-stage. In preferred embodiments, themethods further comprise transferring an embryo into the uterus, theuterine cavity or the fallopian tubes of a female, preferably wherein ablastocyst-stage embryo is transferred.

The disclosure further provides methods of implanting an embryo in afemale subject, comprising transferring an embryo into the uterus, theuterine cavity or the fallopian tubes of a female and administering tothe female an oxytocin receptor antagonist such that the effect of theantagonist overlaps with the blastocyst-stage of the embryo and/or thefemale is in the receptive endometrium stage.

In preferred embodiments, the female is undergoing transfer of ablastocyst-stage embryo and the antagonist is administered to the femalesuch that the antagonist is released to the female on the same day thatthe embryo is transferred. Preferably, the antagonist is administeredbetween 2 hours prior to and 2 hours post embryo transfer (for example,in an immediate release formulation), preferably wherein the antagonistis administered twice, preferably wherein the first administrationoccurs around 45 minutes prior to embryo transfer and the secondadministration occurs around 60 minutes after the first administration.Preferably, a blastocyst-stage embryo has an expansion and hatchingstatus of 3, 4, 5, or 6, more preferably wherein the blastocyst-stageembryo is a day 5 post-insemination embryo.

In preferred embodiments, the female is undergoing transfer of acleavage-stage embryo and the antagonist is administered to the femalesuch that the antagonist is released two or three days after the embryois transferred. Preferably, a cleavage-stage embryo has at least 6blastomeres and fragmentation of 20% or less, preferably wherein thecleavage-stage embryo is a day 2 or day 3 post-fertilization embryo.

In preferred embodiments, the antagonist is a selective oxytocinreceptor antagonist or an vasopressin/oxytocin receptor antagonist. Morepreferably, the antagonist is a selective oxytocin receptor antagonist.

Preferably, the antagonist is barusiban. Preferably, barusiban isprovided subcutaneously. Preferably, between 30-80 mg, more preferably50 mg of barusiban is administered. In some embodiments, the female isundergoing transfer of a blastocyst-stage embryo and barusiban isadministered to the female as a depot prior to the day of embryotransfer. Preferably, the female is undergoing transfer of ablastocyst-stage embryo and barusiban is administered to the female onthe same day as embryo transfer. Preferably, 40 mg barusiban isadministered subcutaneously about 15, 30, 45, 60, or 75 minutes (e.g.,45 minutes) prior to blastocyst stage embryo transfer and 10 mgbarusiban is administered subcutaneously about 15, 30, 45, 60, or 75minutes (e.g., 60 minutes) after the first administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict examples of oxytocin receptor antagonists.

FIG. 2 depicts odds ratio for ongoing implantation rate by day oftransfer for (BASIC) clinical trial.

FIG. 3 is a graph of ongoing implantation rate by day of transfer for(BASIC) clinical trial.

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS

As used herein, “to comprise” and its conjugations is used in itsnon-limiting sense to mean that items following the word are included,but items not specifically mentioned are not excluded. In addition theverb “to consist” may be replaced by “to consist essentially of” meaningthat a compound or adjunct compound as defined herein may compriseadditional component(s) than the ones specifically identified, saidadditional component(s) not altering the unique characteristic of theinvention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The word “approximately” or “about” when used in association with anumerical value (approximately 10, about 10) preferably means that thevalue may be the given value of 10 more or less 1% of the value.

When referring herein to a range, such as, e.g., a range of days, therange includes both end points. For example, day LH+6 to day LH+9encompasses day LH+6, day LH+7, day LH+8, and day LH+9.

As used herein, the term “embryo” refers to a zygote up to eight weeksafter fertilization. “Embryo transfer” is the procedure in which one ormore embryos are placed into the uterus, uterine cavity, or fallopiantubes of a female.

As used herein, a female subject is a mammal which includes humans;companion animals, e.g., dogs and cats; domestic livestock animals, suchas pigs, horses, donkeys, goats, sheep, llamas; as well as rare andthreatened species. Preferably, the subject is human.

Assisted reproductive technology (ART) refers to methods for achievingpregnancy using artificial means. Preferably, ART refers to methods inwhich an in vitro fertilized embryo is transferred into a femalesubject, for example using IVF/ICSI.

“Fresh embryo transfer” refers to the transfer of an embryo withoutfirst freezing the embryo.

Generally, the natural ovulation cycle ranges from 21 to 35 days, withthe average length being 28 days. The first part of the cycle isreferred to as the follicular phase in which the ovarian folliclesmature. Ovulation follows by which a mature egg is released into theoviduct. The luteal phase refers to a phase of the ovulation cyclebeginning with the formation of the corpus luteum at LH+1 and finishingthe day before the first day of menstruation.

“Ongoing implantation rate” as used herein refers to the number ofintrauterine viable fetuses 10-11 weeks after transfer divided by numberof embryos/blastocysts transferred. Preferably, the administration of anoxytocin receptor antagonist as disclosed herein increases the ongoingimplantation rate by at least 5%, more preferably by at least 10% andmost preferred by at least 20%.

“Ongoing pregnancy rate” as used herein refers to a pregnancy with atleast one intrauterine viable fetus 10-11 weeks after transfer dividedby number of embryos/blastocysts transferred. Preferably, theadministration of an oxytocin receptor antagonist as disclosed hereinincreases the ongoing pregnancy rate by at least 5%, more preferably byat least 10% and most preferred by at least 20%.

“Implantation rate” as used herein refers to the number of intrauterinegestational sacs with fetal heart beat 5-6 weeks after transfer dividedby number of embryos/blastocysts transferred. Preferably, theadministration of an oxytocin receptor antagonist as disclosed hereinincreases the ongoing implantation rate by at least 5%, more preferablyby at least 10% and most preferred by at least 20%.

“Clinical pregnancy rate” as used herein refers to a pregnancy with atleast one intrauterine gestational sac with fetal heart beat 5-6 weeksafter transfer divided by number of embryos/blastocysts transferred.Preferably, the administration of an oxytocin receptor antagonist asdisclosed herein increases the clinical pregnancy rate by at least 5%,more preferably by at least 10% and most preferred by at least 20%.

“Live birth rate” refers to the number of live births per women treated.Preferably, the administration of an oxytocin receptor antagonist asdisclosed herein increases the live birth rate by at least 5%, morepreferably by at least 10% and most preferred by at least 20%.

“Therapeutically effective amount”, as used herein, refers to an amountthat produces the desired effect for which it is administered. In someembodiments, the term refers to an amount that is sufficient, whenadministered to a female subject undergoing embryo transfer inaccordance with a therapeutic dosing regimen, to increase ongoingimplantation rate, to increase ongoing pregnancy rate, to increaseclinical pregnancy rate, and/or to increase live birth rate. Those ofordinary skill in the art will appreciate that the term “therapeuticallyeffective amount” does not in fact require successful treatment beachieved in a particular individual. Rather, a therapeutically effectiveamount may be that amount that provides a particular desiredpharmacological response in a significant number of subjects whenadministered to female subjects in need of such treatment. Those ofordinary skill in the art will appreciate that, in some embodiments, atherapeutically effective amount of a particular agent or therapy may beformulated and/or administered in a single dose. In some embodiments, atherapeutically effective agent may be formulated and/or administered ina plurality of doses, for example, as part of a dosing regimen.

One aspect of the disclosure provides oxytocin receptor antagonists foruse in increasing ongoing implantation rate, increasing ongoingpregnancy rate, increasing clinical pregnancy rate, and/or increasinglive birth rate, relative to a control, in a female subject undergoingembryo transfer. Preferably, the ongoing implantation rate is increasedrelative to a control.

Previous large studies in the art report administration of oxytocinreceptor antagonists in the early luteal phase (corresponding to day 2or 3 post-retrieval) when uterine contraction frequency is high. Theexpectation was that these compounds, which reduce uterinecontractility, would improve embryo implantation. However, the oxytocinreceptor antagonists demonstrated no improved effects on implantationwhen provided in the early luteal phase (Ng et al. 2014). Thus, incertain embodiments, the present invention excludes immediate release orsubstantially immediate release formulations of oxytocin receptorantagonists administered in the early luteal phase (i.e., preceding thereceptive endometrium stage).

The present disclosure demonstrates the effectiveness of oxytocinreceptor antagonists on embryo implantation when provided after theearly luteal phase, or rather, at the receptive endometrium stage and/orthe when the embryo has reached the blastocyst-stage (see Example 1).Since the frequency of uterine contractions has returned or nearlyreturned to baseline at this stage, it was surprising and unexpectedthat a oxytocin receptor antagonist had an effect on the implantationrate.

Implantation is a critical process in which an embryo apposes, attachesand invades the endometrium. The uterus will accept the implantingembryo only during a limited period of time described as the “window ofimplantation” or “receptive window” (Makrigiannakis and Minas 2006;Strowitzki et al. 2006). The window of implantation is a period of a fewdays in which the endometrium acquires the receptive stage allowingembryo adhesion and invasion (Koot and Macklon 2013). This stage isreferred to herein as the “receptive endometrium stage”.

Successful implantation depends not only a receptive endometrium, butalso on a functional embryo and the synchronized communication betweenthe embryo and maternal tissues. Therefore, during the receptive windowof implantation, the embryo also needs to be at the appropriate stage.Implantation occurs after a blastocyst hatches from the zona pellucida.Therefore, as is well known if the field of ART, if a blastocyst stageembryo is transferred, the woman should ideally be in the receptiveendometrium stage, so that both the endometrium and the embryo aresynchronized for implantation. If a cleavage stage embryo istransferred, then the woman should be in the pre-receptive stage. Theendometrium and embryo will both further develop such that when theembryo reaches the blastocyst stage, the endometrium will have reachedthe receptive stage.

Accordingly, the antagonists disclosed herein are administered such thatthe effect of the antagonist overlaps with the receptive endometriumstage and/or the embryo reaching the blastocyst-stage. Preferably, theantagonists are provided such that the antagonist is released orcontinues to be released in the receptive endometrium stage and/or theembryo reaching the blastocyst-stage. As discussed further herein, theantagonists are usually formulated as immediate release compositionssuch that they are administered during the receptive endometrium stage.However, the disclosure also encompasses antagonists formulated ascontrol or delayed release formulations, for example as a depot, suchthat they are administered during the pre-receptive stage, but arereleased during the receptive stage.

A number of cellular and morphological changes are associated with thetransformation of a pre-receptive endometrium to a receptiveendometrium. Biomarkers have also been identified which can be used toevaluate whether the endometrium is in a receptive stage. For example,the Endometrial Receptivity Array from Ignomix™ analyzes the expressionof 238 genes in order to determine whether the endometrium is in thereceptive stage (see, WO2010010201 and WO2010010213). Preferably, thereceptive endometrium stage is defined as having a normal receptiveprofile based on the expression profile of one or more (e.g., all) ofthe 238 genes of the Endometrial Receptivity Array (ERA).

The receptive endometrium stage can also be characterized based on thestage of a normal ovulation cycle. Ovulation occurs after theluteinizing hormone (LH) surge, which normally takes place around day 14of a normal ovulation cycle. The precise stage of the ovulation cyclecan be characterized based on the timing of the LH surge. The LH surgecan be measured by taking blood samples at various days of a woman'scycle. The day of the LH surge is considered as day LH 0. LH+1 thenusually corresponds to day 15 of the cycle and LH+7 usually to day 21.The endometrium becomes receptive to implantation at around day LH+7 innatural cycles and remains receptive for usually about 4 days (Bergh andNavot 1992), although this timing varies for each woman. In preferredembodiments, the receptive endometrium stage corresponds to between dayLH+6 and day LH+9 of a natural ovulation cycle, more preferably betweenLH+6 and LH+8. The receptive window lasts normally only 2-3 days perovulation cycle. However, as is well-known in the art, there existsvariability between women in both the length of the window and when itoccurs.

In women undergoing oocyte retrieval for fresh embryo transfer, thereceptive window can be characterized based on the day post-oocyteretrieval, the number of days in luteal phase support following oocyteretrieval, and/or the number of days following hCG administration.

In a typical IVF procedure, ovarian stimulation is used in order tostimulate the ovaries to produce multiple eggs. Gonadatropin releasinghormone (GnRH) agonists and GnRH antagonists can be given to preventpremature ovulation while human menopausal gonadotropin (hMG), folliclestimulating hormone (FSH), luteinizing hormone (LH), and clomiphenecitrate can be given to stimulate the production of multiple eggs.Typically, eight to fourteen days of stimulation are required before theovarian follicles are sufficiently developed. Human chorionicgonadotropin (hCG) is usually then administered to ensure the finalstage of maturation and the eggs are retrieved prior to ovulation,usually around 36 hours after hCG administration. The day of hCGadministration is defined as hCG+O and oocyte retrieval is performed onhCG+2.

In preferred embodiments, the receptive endometrium stage corresponds tobetween day hCG+6 and day hCG+9 (or rather, 4 to 7 days post-oocyteretrieval), preferably between day hCG+6 and day hCG+8.

Egg retrieval is a minor surgical procedure that can be performed, forexample, using transvaginal ultrasound aspiration. The eggs may beinspected microscopically and diagnosed to observe their morphologicalfeatures. Insemination is then performed in vitro, for example byincubating oocytes together with sperm or by intracytoplasmic sperminjection (IC SI) in whichsperm is injected with a microscopic needleinto the egg. “Fertilization” refers to the penetration of the ovum bythe spermatozoa and combination of their genetic material resulting inthe formation of a zygote.

After fertilization, embryos are cultured in vitro. Methods forculturing and staging embryos are well-known in the art and aredescribed in, e.g., US 20140134632, US 20140017717, US 20120252119, andUS 20120252119, which are herein incorporated by reference in theirentirety. Culture media known in the art that are suitable for use forthe in vitro support of cell development and growth include human tubalfluid (HTF) (Irvine Scientific), N-2-hydroxyethylpiperazine-N′-2-ethane(HEPES) media (Irvine Scientific), IVF-50 (Scandanavian IVF Science), S2(Scandanavian IVF Science), Gl and G2 (Scandanavian IVF Science),UnilVF, ISM-1, BlastAssist, UTM media (sold as MEDICULT® media by OrigioA/S), Modified Whittens medium, Wittinghams T6 media, Ham's F-10 media,and Earle's solution. G1 and G2 media were specifically formulated tomeet the physiological needs of the cleavage stage embryo and the embryoin the eight-cell through blastocyst stage of development. U.S. Pat. No.6,605,468 discloses a medium for the propagation of early stage embryosto blastocyst stage.

Embryos may also be subjected to morphological, kinetic and/or genetictesting. Preferably, visual observation of the embryo by microscopy isused to determine if aberrant physical or morphological features arepresent (see, e.g., WO2013078312). Preimplantation genetic diagnosis iscommonly performed to screen for inherited diseases. For this method,one or two cells are removed from an embryo to test for geneticdiseases.

Methods of embryo transfer are well known in the art. One or moreembryos may be aspirated into a catheter and inserted into the uterus,the uterine cavity or the fallopian tubes.

In some embodiments a cleavage-stage embryo is transferred.“Cleavage-stage” embryos range from 2-cells to 16 cells and can becharacterized based on, e.g., fragmentation, symmetry of division, andabsence of multinucleation (see Prados et al. Human Reproduction 201227:50-71 for review). Fragmentation is generally characterized by thepercent of embryo volume that is replaced by fragments. Preferably, acleavage-stage embryo is characterized as having 4 blastomeres on day 2post-insemination and 6-8 blastomers on day 3 post-insemination.Preferably, the cleavage-stage embryo has at least 6 blastomeres andfragmentation of 20% or less.

Preferably, a day 2 or day 3 cleavage stage embryo is transferred. Infresh embryo transfers where the female has undergone oocyte retrieval,the day 2 or day 3 cleavage stage embryo is then usually transferred 2or 3 days post-oocyte retrieval, respectively. The blastocyst stage ofthe embryo and the receptive endometrium stage are reached, ideallysimultaneously, several days after transfer.

Preferably, a blastocyst-stage embryo is transferred. A“blastocyst-stage” embryo has an inner cell mass, an outer cell layercalled the trophectoderm, and a fluid-filled blastocele cavitycontaining the inner cell mass from which the whole of the embryo isderived. An embryo normally reaches this stage at day 5 or 6post-retrieval. A blastocyst-stage embryo can be characterized based onits expansion and hatching status. Expansion relates to the increasingvolume of the cavity (i.e. blastocoel), while hatching refers to theherniation or escape of the blastocyst from its membrane (i.e. zonapellucida). The expansion and hatching status is characterized asfollows:

1. An early blastocyst, blastocoel being less than half volume of thatof the embryo

2. A blastocyst with a blastocoel whose volume is half of, or greaterthan half of, that of the embryo

3. A blastocyst with a blastocoel completely filling the embryo

4. An expanded blastocyst with a blastocoel volume larger than that ofthe early embryo, with a thinning zona

5. A hatching blastocyst with the trophectoderm starting to herniatethrough the zona

6. A hatched blastocyst, in which the blastocyst has completely escapedfrom the zona.

In preferred embodiments, the blastocyst-stage embryo for transfer hasan expansion and hatching status of 3, 4, 5, or 6.

In fresh embryo transfers where the female has undergone oocyteretrieval, the blastocyst stage embryo is usually transferred to thefemale 5 or 6 days post-oocyte retrieval, preferably 5 dayspost-retrieval.

For women undergoing fresh embryo transfer following oocyte retrieval,the endometrium on day 2 and day 3 post-oocyte retrieval is typically ina pre-receptive stage and is not conducive to implantation. In preferredembodiments, the receptive endometrium stage corresponds to between day4 and day 7 post-oocyte retrieval, preferably between day 5 and day 6.If hCG is used to induce ovulation or trigger final maturation, days 4to 7 post-oocyte retrieval will normally correspond to hCG+6 to hCG+9.

Ovarian stimulation with fertility drugs usually leads to luteal phasedeficiency. Therefore, it is generally standard practice for lutealphase support to be used in women following oocyte retrieval. Lutealphase support refers to therapeutic interventions during the lutealphase aiming at supplementing corpus luteal function for improving theembryo implantation and the early pregnancy development. Luteal phasesupport is known in the art and usually comprises supplementation withprogesterone, estradiol and progesterone, progestins, hCG, and/or a GnRHagonist, or rather the administration of exogenous progesterone,estradiol and progesterone, progestins, hCG, and/or a GnRH agonist.Progesterone is normally administered intramuscularly or vaginally,while hCG is administered intra-muscularly or subcutaneously.Preferably, luteal phase support begins the first day after oocyteretrieval, i.e., day 1 post-oocyte retrieval.

Preferably, the receptive endometrium stage corresponds to between day 4and day 7, preferably between day 4 and day 6, of luteal phase supportin women who have undergone oocyte retrieval. In preferred embodiments,the female has undergone ovarian stimulation in preparation for oocyteretrieval.

The present disclosure encompasses the transfer of embryos into a femalewithin a few days post fertilization (i.e., fresh embryo transfer), aswell as the use of frozen embryos. Frozen embryo transfer (FET) is aknown procedure that utilizes cryopreserved embryos from a previouscycle of in vitro fertilization or ICSI. The cryopreserved embryos arethawed and transferred into the uterine cavity through a catheter. Thedisclosure also encompasses the use of cryopreserved oocytes forfertilization. In these embodiments, oocytes can be later thawed,fertilized, and cultured and transferred as described herein.

Rapid freezing can be used for these purposes, for example together witha cryoprotectant. Conventional cryoprotectants include glycols such asethylene glycol, propylene glycol, and glycerol;2-methyl-2,4-pentanediol (MPD); dimethyl sulfoxide (DMSO) and sucrose.Alternatively, vitrification can also be used to freeze oocytes orembryos.

FET, as well as “third-party IVF” (gestational surrogacy, ovum donation,embryo donation), may be performed during a natural ovulation cycle. Thereceptive window for these women can be determined based on a naturalovulation cycle as described herein. In some embodiments, ovulation isinduced with the administration of, e.g., hCG. Preferably, in thesewomen the receptive window corresponds to between hCG+6 and hCG+9,preferably between hCG+6 and hCG+8.

In some embodiments, women undergoing FET or third party IVF alsoreceive luteal support as described above. Preferably in these women thereceptive window corresponds to between day 4 and day 9, preferablybetween day 5 and day 7, of luteal phase support. Luteal phase supportis often used when FET or third party IVF is performed during an“artificial cycle”. In these cases, the endometrium is prepared byadministering estrogen and/or progesterone using methods known in theart. Preferably, luteal phase support begins after the endometrium isprimed for at least 6 days with exogenous estrogen in order to induce anartificial cycle.

In an exemplary embodiment of FET or third party IVF, estrogen isprovided orally or vaginally in doses of 4-8 mg daily for about 10 days,at which time luteal phase support is initiated with the administrationof vaginal progesterone and blastocyst transfer occurs 6 days afterstarting progesterone.

In one embodiment of the disclosure, the effect of the oxytocin receptorantagonist overlaps with the blastocyst-stage of the embryo. Preferably,the antagonist is released when the embryo has reached theblastocyst-stage. Preferably, a blastocyst-stage embryo is transferredto said female and the antagonist is administered on the same day thatthe embryo is transferred.

The disclosure also contemplates the transfer of a cleavage-stageembryo. In these embodiments, the antagonist may be provided as asustained or controlled release formulation for release several daysafter transfer. Alternatively, the antagonist may be administered as animmediate release formulation several days after transfer. As shown inthe examples, administration of barusiban (an oxytocin receptorantagonist) when an embryo is in the blastocyst-stage results in anincrease in ongoing implantation rate from 27% to 45%, a significantincrease.

Oxytocin receptor antagonists are known in the art, and any oxytocinreceptor antagonist can be used in the methods of the disclosure.Preferably, the oxytocin receptor antagonists are selected fromselective oxytocin antagonists and mixed vasopressin/oxytocin receptorantagonists. A selective oxytocin antagonist has a Ki for the oxytocinreceptor which is at least one order of magnitude higher than the Ki forthe vasopressin receptor.

Receptor antagonists include compounds which reduce the expressionand/or activity of an oxytocin receptor. A preferredvasopressin/oxytocin receptor antagonist is atosiban(1-(3-mercaptopropanoicacid)-2-(O-ethyl-D-tyrosine)-4-L-threonine-8-L-ornithine-oxytocin).

Oxytocin receptor antagonists also include RNA interference or RNAantisense molecules directed against said receptor.

Preferably, the antagonist binds to the receptor and prevents receptoractivity. Such antagonists include, e.g., antibodies directed againstthe receptor (e.g., “neutralizing antibodies”) as well as smallmolecules. Preferably, the antagonist acts as a competitive antagonistand competes with oxytocin for binding to the oxytocin receptor.Preferably, the antagonists are small molecules that bind to thereceptor and antagonize receptor activity. A preferred selectiveoxytocin antagonist is barusiban.

Suitable oxytocin receptor antagonists are well-known to the skilledperson and can be easily identified based on known screening methodswhich use, e.g., receptor activation and/or receptor binding as aread-out. Suitable antagonists include those disclosed in U.S. Pat. No.6,143,722, which is hereby incorporated by reference in its entirety.U.S. Pat. No. 6,143,722 discloses heptapeptide analogues, or apharmaceutically acceptable salts thereof, having oxytocin antagonistactivity and consisting of a hexapeptide moiety S and a C-terminalbeta-aminoalcohol residue Z bound to the moiety S by an amide bond,wherein the beta-aminoalcohol Z is:

wherein Q is (CH2)n-NH-A, n is 1-6 and A is H or —C(═NH) NH2,

-   and wherein R is CH3 or C2H5;-   and the moiety S is:

wherein Mpa, Ile, Asn and Abu have the following meaning:

Mpa 3 mercaptopropionic acid residue

Ile isoleucine residue

Asn asparagine residue

Abu alpha-aminobutyric acid residue;

and wherein X is a D-aromatic alpha-amino acid; and Y is an aliphaticalpha-amino acid. Preferred oxytocin antagonists are listed in FIG. 1.

Further antagonists include OBE001/AS-602305 (in particular oralformulations thereof), TT-235 (Northwestern University), the selectiveoxytocin receptor antagonist Epelsiban((3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(2,6-dimethylpyridin-3-yl)-2-(morpholin-4-yl)-2-oxoethyl]-6-[(1S)-1-methylpropyl]piperazine-2,5-dione);Retosiban((3R,6R)-6-[(2S)-butan-2-yl]-3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(2-methyl-1,3-oxazol-4-yl)-2-(morpholin-4-yl)-2-oxoethyl]piperazine-2,5-dione);PF-3274167(5-(3-(3-(2-chloro-4-fluorophenoxy)azetidin-1-yl)-5-(methoxymethyl)-4H-1,2,4-triazol-4-yl)-2-methoxypyridine);and L-368,899 hydrochloride (CAS 148927-60-0); L-371,257(1-[1-[4-(1-acetylpiperidin-4-yl)oxy-2-methoxybenzoyl]piperidin-4-yl]-4H-3,1-benzoxazin-2-one).Additional oxytocin antagonists are also described, e.g., inWO2004020414 and WO2005/028452, which are hereby incorporated byreference in their entirety. Preferably, the antagonist is selected frombarusiban, atosiban, OBE001/AS-602305, PF-3274167 Epelsiban, andretosiban.

Preferably, the oxytoxin receptor antagonist is formulated in apharmaceutical composition. The composition may also includepharmaceutically acceptable additives such as preservatives, diluents,dispersing agents, agents to promote mucosal absorption (examples ofwhich are disclosed by Merkus, F. W. H. M. et al., J. Controlled Release24, 201-208, 1993, and which include surfactants, bile acids, fusidates,phospholipids and cyclodextrins), buffering agents and flavourings. Suchcompositions may be formulated as solids (for example as tablets,capsules or powders) or liquids (for example as solutions orsuspensions), which is here taken to include creams and ointments, fororal or parenteral administration. Oral (including sublingual andbuccal), intranasal, pulmonary, transdermal, rectal, vaginal,subcutaneous, intramuscular and intravenous administration may all besuitable routes for dosing.

In some embodiments, the pharmaceutical composition can be delivered ina sustained or delayed release system. For example, the antagonist maybe administered using a transdermal patch or formulated in lipophilicdepots (e.g. fatty acids, waxes, oils). As used herein, a sustained ordelayed release system ensures that the antagonist is also present inthe subject at a time point after administration, e.g., several hours oreven several days after administration. Such sustained or delayedrelease systems allow the administration of the receptor antagonistsbefore the female is in a receptive endometrium stage. The sustained ordelayed release, however, ensures that a sufficient amount (or rather atherapeutically effective amount) of the antagonist is still presentwhen the female enters the receptive endometrium stage and/or when theembryo has reached the blastocyst-stage.

In embodiments where the female is undergoing transfer of acleavage-stage embryo, the antagonist should be released when thecleavage-stage embryo has developed into a blastocyst-stage embryoand/or when the female has reached the receptive endometrium stage. Ifan immediate release formulation is used, then the antagonist should beadministered several days after embryo transfer, preferably two or threedays after embryo transfer as this will correspond to the time when thecleavage-stage embryo has developed into a blastocyst-stage embryoand/or when the female has reached the receptive endometrium stage. Ifsustained or delayed release formulations are used, these may beadministered earlier, for example on the day of embryo transfer,provided that the antagonist is released when the cleavage-stage embryohas developed into a blastocyst-stage embryo and/or when the female hasreached the receptive endometrium stage.

In embodiments where the female is undergoing transfer of ablastocyst-stage embryo, the antagonist should be released on the sameday as embryo transfer (e.g., within the same 24 hour period), as thiswill correspond to the time when the embryo has reached theblastocyst-stage and the female has reached the receptive endometriumstage. Preferably, the antagonist is administered between 2 hours priorto and 2 hours post embryo transfer. More preferably, the antagonist isadministered twice. In an exemplary embodiment using barusiban, thefirst administration may take place around 45 minutes prior to embryotransfer and the second administration around 60 minutes after the firstadministration.

As is well-known to a skilled person, the timing of administration isdependent of the particular antagonist used, in particular on thehalf-life of the antagonist. Antagonists with relatively shorthalf-lives may need to be administered multiple times in order to ensurethat their effects overlap the blastocyst-stage and/or the receptiveendometrium stage.

REFERENCES

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Lesny P, Killick S R, Tetlow R L, Robinson J, Maguiness S D. Embryotransfer—can we learn anything new from the observation of junctionalzone contractions? Hum Reprod 1998; 13: 1540-1546.

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All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The invention is further explained in the following examples. Theseexamples do not limit the scope of the invention, but merely serve toclarify the invention.

EXAMPLES Example 1

A randomized, placebo-controlled, double-blind, parallel groups,multinational, multicenter trial assessing the effect of barusibanadministered subcutaneously on the day of transfer on implantation andpregnancy rates in IVF/ICSI patients

Methodology

BASIC was a randomized, double-blind, placebo-controlled, parallelgroups, multinational, multicenter trial. It was designed to evaluatethe effects of barusiban, administered either on the day ofcleavage-stage embryo transfer or on the day of blastocyst transfer, onongoing implantation rate in IVF/ICSI patients. The patients underwentcontrolled ovarian stimulation in the long GnRH agonist or GnRHantagonist protocol, received hCG for triggering of final follicularmaturation, had undergone oocyte retrieval, and had daily luteal phasesupport by supplementation with vaginal progesterone starting on day 1post-retrieval, and transfer on day 3 or 5 post-retrieval. Patients wererandomized in a 1:1 ratio to either the barusiban group or the placebogroup, with stratification according to the day of transfer (day 3post-retrieval or day 5 post-retrieval) and the number ofembryos/blastocysts to be transferred (1 or 2). In total, 255 IVF/ICSIpatients were randomized in the trial and contributing with 440embryos/blastocysts.

Investigational medicinal product, i.e., barusiban or placebo accordingto randomization, was administered as subcutaneous injections at twotime points: 1st administration of 40 mg barusiban or placebo was 45 minprior to transfer and the 2nd administration of 10 mg barusiban orplacebo was 60 min after the 1st administration.

Transfer was performed on day 3 (cleavage-stage embryos) or day 5(blastocysts) after oocyte retrieval. On day 3, only embryos of goodquality defined as ≧6 blastomeres and ≦20% fragmentation weretransferred. On day 5, blastocysts with expansion and hatching status 3,4, 5 or 6 were transferred. The actual number of transferredembryos/blastocysts for each individual patient depended on theavailability of embryos/blastocysts of the required morphologicalquality, local regulations and clinical practice for the patient's age,but the maximum number was 2.

Key aspects related to the transfer procedure had been standardized. Aspeculum was inserted into the vagina and cleaning of the vagina andcervix was done according to local practices but with minimalmanipulation and disturbance. Soft or ultrasoft catheters were used. Theouter sheath of the catheter was inserted just protruding to theinternal os (i.e. keeping the outer sheath in the cervical canal). Theembryo(s)/blastocyst(s) were loaded into the inner sheath which was theninserted through the outer sheath. Using abdominal ultrasound guidance,the embryo(s)/blastocyst(s) were placed 1.5-2.0 cm from the fundus. Thetime from loading the inner catheter to placing theembryo(s)/blastocyst(s) should not have exceeded 1 min. After placement,the inner and outer catheters were withdrawn and checked for retainedembryo(s)/blastocyst(s), mucus and blood. After confirmation that therewere no embryo(s)/blastocyst(s) left in the catheters, the speculum wassubsequently removed; this occurred approximately within 2 min afterplacement of the embryo(s)/blastocyst(s). Any difficulties/eventualitiesoccurring during the transfer procedure were recorded.

Patients received vaginal progesterone tablets 100 mg twice daily fromthe day after oocyte retrieval and until the day of the clinicalpregnancy visit. On the day of transfer, patients should insert theprogesterone tablets at least 3 hours before transfer and at least 3hours after transfer. Ongoing implantation rate (primary endpoint) wasdefined as the number of intrauterine viable fetuses 10-11 weeks aftertransfer divided by number of embryos/blastocysts transferred.

With respect to statistical methodology, the primary hypothesis wastested using a logistic regression model with ongoing implantation(yes/no) as the outcome and treatment and randomization strata asfactors. The treatment effect is presented on the odds ratio scale, asthis represents the outcome of the logistic regression analysis; ananalysis that allows for inclusion of factors and co-variates. It mustbe stressed that the odds ratio based on the logistic regression modelprovides the most appropriate way of representing the data and the basisfor evaluation of treatment effect.

Several factors impact the probability of a successful outcome of anembryo transfer, including the day of transfer, the number ofembryos/blastocyst transferred, and the quality of the embryo/blastocysttransferred. The randomization was stratified for the first two of thesefactors ensuring comparable groups. However, as the embryos developcontinuously it was not feasible to stratify for the quality of thetransferred embryo/blastocyst. Instead, adjustment for the quality ofthe embryo/blastocyst was included in the primary analysis.

Results

The impact of treatment on the different receptivity stages andimplantation potential of cleavage-stage embryos and blastocysts wasapparent, as illustrated by overall ongoing implantation rates of 19%for day 3 post-retrieval transfers and 38% for day 5 post-retrievaltransfers. The same pattern was observed in both the barusiban andplacebo groups.

The observed overall (day 3+day 5) ongoing implantation rate in thetrial was 27.1% for barusiban and 28.2%¹, corresponding to an oddsratio² of 1.1 (95% confidence interval 0.7-1.8; p=0.6960), i.e., infavor of barusiban but not significant. Therefore, the primary endpointfor the overall trial population was not met, but as described belowthis was because the day of transfer had an interaction. Transfer ofcleavage-stage embryos on day 3 post-retrieval yielded an odds ratio of0.3 (0.3-1.2; p=0.1509) (FIG. 2). Analysis of the day 5 post-retrievalstrata resulted in an odds ratio of 2.3 (1.1-4.7; p=0.0270) and therebydemonstrated a significant treatment effect of barusiban on ongoingimplantation rate for blastocyst transfers (FIG. 2). An odds ratio of2.3 corresponds to adjusted means of ongoing implantation rates forblastocyst transfers of 45% for barusiban vs 27% for placebo (relative Aof 67%) (FIG. 3). ¹ Data presented in this document are for theper-protocol (PP) population Similar results were observed for theintention-to-treat (ITT) population. For example, the ongoingimplantation rate for the ITT population was 26.2% for barusiban and27.9% for placebo.² The odds ratios are based on the logistic regressionmodel, for which the analyses are adjusted for site, primary reason forinfertility and embryo/blastocyst quality.

The results from the BASIC trial indicated that interpretation of theeffects of an oxytocin antagonist on implantation rate was affected byday of transfer; cleavage-stage embryo transfer (day 3 post-retrieval)or blastocyst transfer (day 5 post-retrieval). No effect on ongoingimplantation rate was established for barusiban when cleavage-stageembryo transfer was done on day 3 (pre-receptive stage). However, asignificant (p=0.0270) effect of barusiban on improving ongoingimplantation rate was observed when blastocyst transfer was done on day5 (receptive stage).

The BASIC trial identified the time window for a clinically relevantimpact of barusiban, or generally for oxytocin antagonists and mixedoxytocin/vasopressin antagonists, on implantation, which was notpredicted in advance of the trial. An effect on implantation rate isseen when the oxytocin antagonist is administered at the time ofimplantation, on day 5 post-retrieval (or later), but not in the earlyluteal phase on day 2-3 post-retrieval.

The lack of a consistent effect between day 3 post-retrievalcleavage-stage embryo transfers and day 5 post-retrieval blastocysttransfers is of importance for the hypotheses on the mechanisms relatedto uterine contractility and consequences for cycle outcome. For bothdays, the dose and method of administration of pharmacologicalintervention as well as the transfer procedure were the same. Therefore,the mechanisms proposed such as expulsion or dispersion ofembryos/blastocysts due to uterine contractility in the early lutealphase or due to the embryo/blastocyst transfer procedure should nolonger be considered potential explanations for improvement ofimplantation rates with utero-relaxant agents, including oxytocinantagonists or mixed oxytocin/vasopressin antagonists. In case thesemechanisms were the most relevant, the findings should have been thesame for day 3 and day 5 post-retrieval observations.

1-30. (canceled)
 31. A method of treating a female undergoing embryotransfer, comprising: administering to the female luteal phase support,and administering to the female an oxytocin receptor antagonist: (1)only during a time a) between day 6 following luteinizing hormone (LH)surge (LH+6) and day 9 following LH surge (LH+9) of a natural ovulationcycle; b) between day 6 following human chorionic gonadotropin (hCG)administration (hCG+6) and day 9 following hCG administration (hCG+9) ofan induced ovulation cycle; c) between day 6 and day 7 of a luteal phasesupport following oocyte retrieval; or d) between day 6 and day 9 of aluteal phase support; or (2) only on the day of transfer of an embryo,wherein the transferred embryo is a blastocyst-stage embryo.
 32. Amethod of treating a female undergoing embryo transfer, comprisingadministering to the female: (i) luteal phase support; and (ii) anoxytocin receptor antagonist only during a time a) between day 6 and day7 of the luteal phase support following oocyte retrieval; or b) betweenday 6 and day 9 of the luteal phase support.
 33. The method of claim 31,wherein the luteal phase support comprises progesterone.
 34. The methodof claim 33, wherein the luteal phase support comprises about 100 mgprogesterone.
 35. The method of claim 33, wherein the luteal phasesupport comprises administration of progesterone at least twice daily.36. The method of claim 33, further comprising transferring an embryointo the uterus, the uterine cavity, or the fallopian tubes of thefemale.
 37. The method of claim 36, wherein the luteal phase supportcomprises progesterone administration at least 3 hours before the embryotransfer or at least 3 hours after the embryo transfer.
 38. The methodof claim 33, wherein the progesterone is administered intramuscularly orvaginally.
 39. A method of treating a female undergoing embryo transfercomprising administering to the female an oxytocin receptor antagonist(1) only during a time a) between day 6 following luteinizing hormone(LH) surge (LH+6) and day 9 following LH surge (LH+9) of a naturalovulation cycle; b) between day 6 following human chorionic gonadotropin(hCG) administration (hCG+6) and day 9 following hCG administration(hCG+9) of an induced ovulation cycle; c) between day 6 and day 7 of aluteal phase support following oocyte retrieval; or d) between day 6 andday 9 of a luteal phase support; or (2) only on the day of transfer ofan embryo, wherein the transferred embryo is a blastocyst-stage embryo,and wherein the antagonist is barusiban, atosiban, OBE001/AS-602305,TT-235, Epelsiban((3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(2,6-dimethylpyridin-3-yl)-2-(morpholin-4-yl)-2-oxoethyl]-6-[(1S)-1-methylpropyl]piperazine-2,5-dione);Retosiban((3R,6R)-6-[(2S)-butan-2-yl]-3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(2-methyl-1,3-oxazol-4-yl)-2-(morpholin-4-yl)-2-oxoethyl]piperazine-2,5-dione);PF-3274167(5-(3-(3-(2-chloro-4-fluorophenoxy)azetidin-1-yl)-5-(methoxymethyl)-4H-1,2,4-triazol-4-yl)-2-methoxypyridine);L-368,899 hydrochloride; or L-371,257(1-[1-[4-(1-acetylpiperidin-4-yl)oxy-2-methoxybenzoyl]piperidin-4-yl]-4H-3,1-benzoxazin-2-one).40. The method of claim 39, further comprising transferring an embryointo the uterus, the uterine cavity or the fallopian tubes of thefemale.
 41. The method of claim 39, wherein the antagonist isadministered to the female at day 5 post oocyte retrieval.
 42. Themethod of claim 31, wherein the transferred embryo is a cleavage-stageembryo and the antagonist is administered to the female only during atime a) between day 6 following luteinizing hormone (LH) surge (LH+6)and day 9 following LH surge (LH+9) of a natural ovulation cycle; b)between day 6 following human chorionic gonadotropin (hCG)administration (hCG+6) and day 9 following hCG administration (hCG+9) ofan induced ovulation cycle; c) between day 6 and day 7 of a luteal phasesupport following oocyte retrieval; or d) between day 6 and day 9 of aluteal phase support.
 43. The method of claim 42, wherein thecleavage-stage embryo is a day 2 or day 3 post-fertilization embryo. 44.The method of claim 31, wherein the blastocyst-stage embryo has anexpansion and hatching status of 3, 4, 5, or
 6. 45. The method of claim36, wherein the antagonist is administered to the female between 2 hoursprior to and 2 hours after embryo transfer.
 46. The method of claim 39,wherein the antagonist is administered to the female between 2 hoursprior to and 2 hours after the embryo transfer.
 47. The method of claim31, wherein a blastocyst-stage embryo is transferred to the female, andwherein the antagonist is administered to the female on the same day asthe embryo transfer.